The requirements for bone repair, whether provided by the host or within an implantable tissue-engineered construct, include an extracellular matrix scaffold, cells, a functional vascular supply, and osteoinductive factors. Of these essential elements, vascular supply has been the least studied in the context of tissue engineering in part due to the difficulty in quantifying 3-D vascular structures within tissues. Traditionally, 2-D histological analysis has been used to assess vessel density. However, this approach is semi-quantitative at best and does not easily allow analysis throughout the tissue. High-resolution 3-D microcomputed tomography (micro-CT) imaging coupled with contrast agent perfusion has the potential to overcome these limitations to quantify vascular growth. The recent development of in vivo micro-CT systems has further provided the opportunity to non-invasively monitor mineralized matrix formation within a bone defect in vivo. The goal of this application is to combine and extend these methodologies to better understand the temporal and spatial relationships between vascularization and mineralization in a well defined in vivo bone tissue engineering model. The Specific Aims are to: I. Quantify 3-D vascular growth and mineralized matrix formation within scaffolds implanted into critically-sized segmental bone defects, II. Analyze the influence of porous scaffold architecture on vascular invasion and mineralization, and III. Test the effect of adding a cellular component to implanted constructs on vascularization and mineralization during segmental defect repair. The proposed research is highly significant because it integrates quantitative 3-D imaging techniques with a well characterized in vivo model to better understand the inter-relationship between two processes essential to bone repair: vascularization and mineralization. Lack of a vigorous vascular response may be an important mechanism of delayed or failed bone repair. Decoupling of vascularization and mineralization responses is also possible during for example fibrous tissue repair. The restoration of a functional vascular supply is a critical issue for engineering the repair of a wide variety of tissues in addition to bone. Thus, the proposed studies will establish a valuable new approach for assessing the integration of tissue-engineered constructs into living systems. Furthermore, the developed methodologies will have broad applicability to other research areas, including for example studies on fracture healing, skeletal development, vascular injury, and tumorigenesis.